Physico - Chemical characterization of a bacterial encapsulin nanocage - Poster presentation

Understanding the physico-chemical and structural properties of proteins involved in human pathologies is fundamental for identifying new strategies to counteract disease mechanisms. Among these proteins, encapsulins represent a fascinating class of self-assembling protein nanocarriers of prokaryotic origin, characterized by unique structural and functional properties. These nanocompartments can encapsulate specific cargo proteins both in vivo and in vitro, and they are also capable of binding and sequestering metal ions, a feature that is essential for their physiological role and for a wide range of potential biotechnological and biomedical applications.The assembly of encapsulins into nanocages is a highly regulated process driven not only by the correct folding of individual polypeptide chains, but also by a complex network of non-covalent interactions—hydrogen bonds, hydrophobic contacts, and electrostatic interactions—occurring both within subunits and at the inter-subunit interfaces. Understanding the thermodynamic basis of this process is crucial to elucidate how encapsulins achieve their remarkable structural stability and how they respond to environmental or chemical stimuli.To explore these aspects, Differential Scanning Calorimetry (DSC) experiments were carried out to investigate the folding, stability, and self-assembly properties of a bacterial encapsulin. DSC provides precise measurements of heat changes associated with protein unfolding, thus offering direct insight into thermal stability, folding energetics, and the cooperative transitions that accompany protein denaturation. This approach allows the identification of intermediate states and provides a quantitative description of the energy landscape underlying the folding–unfolding equilibrium.Our DSC results revealed that the thermal denaturation of the encapsulin is not a simple two-state process but rather exhibits a multistep behavior, suggesting the presence of distinct structural domains or cooperative units that unfold independently. This finding is consistent with the modular architecture of encapsulins and their intrinsic capacity to undergo reversible conformational rearrangements.In addition, complementary biophysical techniques were employed to characterize the disassembly and reassembly of the encapsulin nanocage under controlled conditions. Fluorescence spectroscopy, circular dichroism (CD), and UV-visible spectroscopy were used to monitor changes in the secondary and tertiary structures, while Dynamic Light Scattering (DLS) measurements provided information on particle size, homogeneity, and aggregation state. The integration of these techniques enabled a comprehensive analysis of the conformational stability and structural dynamics of the encapsulin system.Overall, this study provides new insights into the molecular determinants governing encapsulin stability, assembly, and interaction with metal ions and cargo proteins. Understanding these mechanisms is essential for elucidating their natural biological role and for exploiting encapsulins as versatile nanoplatforms for targeted delivery, enzymatic confinement, or biosensing applications. The knowledge gained here lays the groundwork for the design of innovative therapeutic and biotechnological strategies that harness the structural robustness and functional adaptability of these remarkable protein nanocages.